The goal of the proposed project is to learn how the Yersinia pestis (plague) secreted protein YopM promotes virulence. The studies also will apply to other pathogenic Yersinia and will facilitate characterization of virulence-related proteins of other important human pathogens that express proteins related to YopM. Moreover, studies of YopM should provide new insights into general cell biology. In Aims 1 and 2, the trafficking pathways of YopM will be mapped, resulting in the identification of proteins and structures to which YopM binds in the cytoplasm and nucleus. The molecule to which YopM binds will be identified by N-terminal analysis of crosslinked and immunoprecipitated YopM partners. In Aim 3, regions of YopM will be identified that are involved in interactions with different eukaryotic molecules, using blocking monoclonal antibodies and peptides. The target cells for YopM will be identified by separating infected splenocytes and employing enhanced chemiluminescence. The relative importance of YopM's interactions will be determined by measuring the virulence of mutants of Y. pestis whose YopM is defective in intracellular trafficking or entry into the nucleus. The studies described in this proposal will reveal the pathways of interactions in relevant host cells, will identify the features of YopM involved in these interactions and will identify the targets of YopM within the host cell.